SUCCESSFUL RECOVERY OF THE NORMAL ELECTROPHYSIOLOGICAL PROPERTIES OF PORB (CLASS-3) PORIN FROM NEISSERIA-MENINGITIDIS AFTER EXPRESSION IN ESCHERICHIA-COLI AND RENATURATION

Neisseria meningitidis PorB class 3 porins obtained either from native membranes (wild-type) or recovered from inclusion bodies following ex pression in Escherichia coli (recombinant), have been reconstituted in to solvent-free planar phospholipid membranes. The wild-type and recom binant porins exhibited the same single-trimer conductance (1-1.3 nS i n 200 mM NaCl), tri-level closure pattern, characteristic of functiona l channel trimers, and pattern of insertion into planar membranes. Bot h proteins were open at low voltages and displayed two voltage-depende nt closure processes, one at positive and the other at negative potent ials, Both showed asymmetric voltage dependence such that one gating p rocess occurred at lower voltages (V-0 = 15 mV) than the other (V-0 = 25 mV). The sign of the potential that resulted in closure at low volt ages varied from membrane to membrane indicating that they map have th e property of auto-directed insertion (in analogy to the mitochondrial channel, VDAC). In the case of the recombinant porin, the steepness o f the voltage dependence of one,eating process was slightly less (n = 1.3) than that observed for the other process or for the wild-type cha nnel (n = 1.5-1.7). Both channels have a high (40%) probability of clo sure even at 0 mV. While both channels show a slight selectivity for C l- over Na+, the selectivity of the recombinant porin is a bit higher (permeability ratio of 2.8 vs. 1.6) as measured using a 2-fold salt gr adient. Thus, the method employed to refold the recombinant porin was successful in not only restoring wild-type structure [H.L. Qi, J.Y. Ta i, M.S, Blake, Expression of large amounts of Neisserial porin protein s in Escherichia coli and refolding of the proteins Into native trimer s, Infect. Immun. 62 (1994) 2432-2439; C.A.S.A. Minetti, J.Y. Tai, M.S . Blake, J.K. Pullen, S.M. Liang, D.P. Remeta, Structural and function al characterization of a recombinant PorB class 2 protein from Neisser ia meningitidis. Conformational stability and porin activity, J. Biol. Chem. 272 (1997) 10710-10720] but also the overall electrophysiologic al function. (C) 1998 Elsevier Science B.V.